Levodopa prodrug mesylate hydrate

ABSTRACT

Crystalline (2R)-2-phenylcarbonyloxypropyl (2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate, methods of making the hydrate, pharmaceutical compositions containing the hydrate, and methods of using the hydrate to treat diseases or disorders such as Parkinson&#39;s disease are provided.

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 61/106,937 filed on Oct. 20, 2008,which is incorporated by reference in its entirety.

Disclosed herein is a crystalline hydrate of a levodopa prodrug mesylatesalt, pharmaceutical compositions containing the hydrate, methods ofmaking the hydrate, and the use of the levodopa prodrug mesylate,hydrate for treating diseases or disorders such as Parkinson's disease.

Parkinson's disease is a disabling, progressive illness that affects onein 1,000 people and generally occurs in people over the age of 50 years.Patients with Parkinson's disease have a deficiency of theneurotransmitter dopamine in the brain as a result of nigrostriatalpathway disruption caused by degeneration of the substantia nigra.Levodopa (L-dopa or L-3,4-dihydroxyphenylalanine), an immediateprecursor of dopamine, is the most commonly prescribed drug fortreatment of this disease.

Following oral administration, levodopa is rapidly absorbed via an aminoacid transporter present in the upper small intestine. Due to the narrowdistribution of this transporter system, the window available forlevodopa absorption is limited and the extent of absorption can dependon the rate at which the drug passes through the upper gastrointestinaltract. Intestinal metabolism of levodopa is the major source of firstpass loss of the drug. Approximately 35% of an administered dose oflevodopa reaches the systemic circulation as intact levodopa after oraladministration in patients (Sasahara, J. Pharm. Sci 1990, 69, 261). Onceabsorbed, levodopa is rapidly metabolized to dopamine by L-aromaticamino acid decarboxylase (AADC) enzymes in the peripheral tissues (e.g.,intestines and liver). For this reason, levodopa is normallyco-administered with a decarboxylase enzyme inhibitor such as carbidopaor benserazide. When administered with carbidopa, the plasmaconcentration of intact levodopa increases and thus more levodopabecomes available to be transported into the central nervous systemwhere it is converted to dopamine. Carbidopa and benserazide do notcross the blood-brain barrier to a significant extent and therefore donot inhibit the required conversion of levodopa to dopamine in thebrain.

Levodopa prodrugs designed to be absorbed from both the small and largeintestines have been described in Xiang et al., U.S. Pat. No. 7,323,585,U.S. Pat. No. 7,342,131, U.S. Patent Application Publication No.2008/0103200, U.S. Pat. No. 7,534,813, U.S. Patent ApplicationPublication No. 2008/0171789, and U.S. Patent Application PublicationNo. 2008/0214663, each of which is incorporated by reference in itsentirety. These levodopa prodrugs can achieve an oral bioavailability oflevodopa that is at least two times greater than the oralbioavailability of levodopa when orally administered on an equivalentmolar basis. More specifically, Xiang et al., U.S. Pat. No. 7,342,131and U.S. Pat. No. 7,534,813 disclose the compound(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate hydrochloride in bothamorphous and crystalline forms (see Example 8 of Xiang et al.).Crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1:

is described by Xiang et al., U.S. Pat. No. 7,563,821. The prodrugsdescribed by Xiang et al. can be efficaciously incorporated intosustained release formulations to provide sustained systemic exposure tolevodopa upon oral administration to a patient.

A crystalline hydrate of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate has beencharacterized.

In a first aspect, the compound (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isprovided.

In a second aspect, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isprovided.

In a third aspect, pharmaceutical compositions comprising at least onepharmaceutically acceptable vehicle and a therapeutically effectiveamount of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orcrystalline form thereof are provided.

In a fourth aspect, methods of treating a disease in a patientcomprising administering to a patient in need of such treatment atherapeutically effective amount of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orcrystalline form thereof are provided.

In a fifth aspect, methods of synthesizing(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate areprovided comprising processing (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate by high shearwet granulation using a water content ranging from about 10 wt-% toabout 20 wt-% to provide granules comprising(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

In a sixth aspect, methods of synthesizing(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate areprovided comprising dissolving (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate in a solventcomprising water to provide a solution; wherein the water activity inthe solvent is greater than 0.6 and crystallizing(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

In a seventh aspect, methods of modulating dopamine levels in a patientare provided comprising administering to the patient a pharmaceuticalcomposition comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate and apharmaceutically acceptable vehicle.

In an eighth aspect, oral dosage forms are provided comprising(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

These and other features provided by the present disclosure are setforth herein.

The skilled artisan will understand that the drawings, described herein,are for illustration purposes only. The drawings are not intended tolimit the scope provided by the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows thermogravimetric scans of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (solid line) and(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate (dashedline).

FIG. 2 shows differential scanning calorimetry scans of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (dashed line)and (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate (solidline).

FIG. 3 shows the sorption and desorption of water from(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate atabout 25° C. as a function of percent relative humidity (% RH).

FIG. 4 shows X-ray powder diffraction patterns of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (patterns 2 and3), (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateprepared by crystallization from isopropanol/water (40/60) (pattern 1),and for dry granules prepared by high shear wet granulation (pattern 4).

FIG. 5 shows X-ray powder diffraction patterns of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (patterns 1-6)or (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate(patterns 7-9) crystallized from isopropanol/water mixtures containingdifferent amounts of water.

FIG. 6 shows differential scanning calorimetry curves for(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateobtained with a temperature ramp of 2° C./min (scan 2) or 20° C./min(scan 1).

FIG. 7 shows X-ray powder diffraction patterns of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate before(pattern 1) and after (pattern 2) heating at 70° C., and after exposureto room temperature (25° C.) and humidity (40-60% RH) for 1 hour(pattern 3) and for 24 hours (pattern 4).

FIG. 8 shows X-ray powder diffraction patterns of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (pattern 3) and(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate before(pattern 2) and after overnight vacuum drying and exposure to roomtemperature (25° C.) and humidity (40-60% RH) for 1 day (pattern 1).

FIG. 9 shows a dissolution profile for sustained release oral tabledosage forms comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

FIG. 10 shows the molecular structure and conformation of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate.

FIG. 11 shows the layered crystal structure of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate.

FIG. 12 shows the asymmetric unit lattice of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate showingtwo symmetry independent drug molecules, mesylate anions and threesymmetry independent water molecules.

FIG. 13 shows the layered crystal structure of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

“Bioavailability” refers to the amount of a drug that reaches thesystemic circulation of a patient following administration of the drugor prodrug thereof to the patient and may be determined by evaluating,for example, the plasma or blood concentration-versus-time profile forthe drug. Parameters useful in characterizing a plasma or bloodconcentration-versus-time curve include the area under the curve (AUC),the time to maximum concentration (T_(max)), and the maximum drugconcentration (C_(max)), where C_(max) is the maximum concentration of adrug in the plasma or blood of a patient following administration of adose of the drug or prodrug thereof to the patient, and T_(max) is thetime to the maximum concentration (C_(max)) of a drug in the plasma orblood of a patient following administration of a dose of the drug orprodrug thereof to the patient.

As used herein, the abbreviation “b.v.” or “bv” means “by volume”.Particularly, when referencing a mixture of more than one fluid, theterm % b.v. means the percentage of one fluid in the total volume. As anon-limiting example a mixture of methanol and water that is 10% b.v.water comprises 10 units of water and 90 units of methanol.

“Crystalline” means having a regularly repeating arrangement ofmolecules, which is maintained over long range or external face planes.

“Compositional purity” in reference to (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate meansthe percent of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate in acomposition relative to the total amount of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate in thecomposition.

“Disease” refers to a disease, disorder, condition, symptom, orindication.

“Hydrate” means associated with water.

“Hydrate of crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate” or “crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate” refersto a crystalline compound in which anhydrous(2R)-2-phenylcarbonyloxypropyl(2R)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate is hydrated withone or more water molecules, including fractional water molecules. Forexample, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate hydrate maycontain from about 1 to about 2 moles of water, including fractionalmoles of water, per mole of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate. In certainembodiments, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateincorporates from about 1.3 to about 1.7 moles of water, per mole of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, and in certainembodiments, about 1.5 moles of water per mole of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (i.e.,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate,sesqui-hydrate).

In certain embodiments, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate maycontain from about 5.1 wt-% to about 5.9 wt-% water, in certainembodiments, from about 5.3 wt-% to about 5.7 wt-% water and in certainembodiments, about 5.5 wt-% water (i.e., (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate,sesqui-hydrate).

“Diastereomeric purity” refers to the percent of one diastereomer of acompound relative to all other diastereomers of the compound in acomposition containing more than one diastereomer of the compound. Forexample, a composition is indicated as having a diastereomeric purity ofabout 97% of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate whenabout 97% of the2-phenylcarbonyloxypropyl-2-amino-3-(3,4-dihydroxyphenyl)propanoatemesylate, hydrate in the composition is the(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratediastereomer and about 3% of the2-phenylcarbonyloxypropyl-2-amino-3-(3,4-dihydroxyphenyl)propanoatemesylate, hydrate in the composition comprises one or more of the otherisomers such as the (2R)-(2R)-, the (2S)-(2R)-, and/or the(2S)-(2S)-isomers. In certain embodiments, the diastereomeric purity ofa composition is, for example, greater than or at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

“Levodopa prodrug mesylate” refers to (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and crystallineform thereof.

“Levodopa prodrug mesylate, hydrate” refers to(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate andcrystalline form thereof.

“Parkinson's disease” is a clinical syndrome comprising bradykinesia(slowness and poverty of movement), muscular rigidity, resting tremor(which usually abates during voluntary movement), and an impairment ofpostural balance leading to disturbance of gait and falling. Othersymptoms include gait and posture disturbances such as shuffling,decreased arm swing, turning “en bloc,” stooped, forward-reflexedposture, festination, gait freezing and dystonia; speech and swallowingdisturbances such as hypophonia, festinating speech, drooling, non-motorcauses of speech/language disturbance in both expressive and receptivelanguage, and dysphagia; as well as fatigue, masked facies,micorpgraphia, impaired fine motor dexterity and coordination, impairedgross motor coordination, and poverty of movement. Non-motor mooddisturbances associated with Parkinson's disease include mooddisturbances such as depression; cognitive disturbances such as slowedreaction time, executive dysfunction, dementia, memory loss, andmedication effects; sleep disturbances such as excessive daytimesomnolence, insomnia, and disturbances in REM sleep; sensationdisturbances such as impair visual perception, dizziness and fainting,impaired proprioception, reduction or loss of sense of smell, and pain;and autonomic disturbances such as oily skin and seborrheic dermatitis,urinary incontinence, constipation and gastric dysmotility, alteredsexual function, and weight loss. The Unified Parkinson's disease Ratingscale is the primary clinical tool used for the diagnosis of Parkinson'sdisease.

“Patient” includes animals and mammals, for example humans.

“Pharmaceutical composition” refers to a composition comprising(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orcrystalline form thereof and at least one pharmaceutically acceptablevehicle with which (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isadministered to a patient. A pharmaceutical composition may furthercomprise anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate or crystallineform thereof.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of a federal or a state government, listed in the U.S.Pharmacopoeia, or listed in other generally recognized pharmacopoeia foruse in mammals, including humans.

“Pharmaceutically acceptable vehicle” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable excipient, a pharmaceutically acceptablecarrier, or a combination of any of the foregoing with which(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orcrystalline form thereof can be administered to a patient and which doesnot destroy the pharmacological activity thereof and which is nontoxicwhen administered in doses sufficient to provide a therapeuticallyeffective amount of the compound.

“Prodrug” refers to a derivative of a drug molecule that requires atransformation within the body to release the active drug. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the parent drug. A carboxyl-containing drug may beconverted to, for example, an ester of either simple alkyl oracyloxyalkyl prodrug, which may be hydrolyzed in vivo to provide thecarboxyl-containing drug. Prodrugs for drugs with functional groupsdifferent than those listed above are well known to those skilled in theart. (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate is aprodrug of L-dopa (levodopa).

“Promoiety” refers to a group bonded to a drug, typically to afunctional group of the drug, via bond(s) that are cleavable underspecified conditions of use. The bond(s) between the drug and promoietymay be cleaved by enzymatic or non-enzymatic means. Under the conditionsof use, for example following administration to a patient, the bond(s)between the drug and promoiety may be cleaved to release the parentdrug. The cleavage of the promoiety may proceed spontaneously, such asvia a hydrolysis reaction, or may be catalyzed or induced by anotheragent, such as by an enzyme, by light, by acid, or by a change of orexposure to a physical or environmental parameter, such as a change oftemperature, pH, etc. The agent may be endogenous to the conditions ofuse, such as an enzyme present in the systemic circulation to which theprodrug is administered or the acidic conditions of the stomach or theagent may be

“Treating” or “treatment” of any disease or disorder refers to arrestingor ameliorating a disease, disorder, or at least one of the clinicalsymptoms of a disease or disorder, reducing the risk of acquiring adisease, disorder, or at least one of the clinical symptoms of a diseaseor disorder, reducing the development of a disease, disorder or at leastone of the clinical symptoms of the disease or disorder, or reducing therisk of developing a disease or disorder or at least one of the clinicalsymptoms of a disease or disorder. “Treating” or “treatment” also refersto inhibiting the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both, and to inhibiting atleast one physical parameter which may or may not be discernible to thepatient. In certain embodiments, “treating” or “treatment” refers todelaying the onset of the disease or disorder or at least one or moresymptoms thereof in a patient which may be exposed to or predisposed toa disease or disorder even though that patient does not yet experienceor display symptoms of the disease or disorder.

“Therapeutically effective amount” refers to the amount of a compoundthat, when administered to a subject for treating a disease or disorder,or at least one of the clinical symptoms of a disease or disorder, issufficient to affect such treatment of the disease, disorder, orsymptom. A “therapeutically effective amount” can vary depending, forexample, on the compound, the disease, disorder, and/or symptoms of thedisease or disorder, severity of the disease, disorder, and/or symptomsof the disease or disorder, the age, weight, and/or health of thepatient to be treated, and the judgment of the prescribing physician. Anappropriate amount in any given instance can be readily ascertained bythose skilled in the art or capable of determination by routineexperimentation.

Reference is now be made in detail to certain embodiments of compounds,compositions, and methods. The disclosed embodiments are not intended tobe limiting of the claims. To the contrary, the claims are intended tocover all alternatives, modifications, and equivalents of the disclosedembodiments.

The levodopa prodrug, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1:

and crystalline form thereof are disclosed by Xiang et al., U.S. Pat.No. 7,563,821. Anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 can beprepared via synthetic methods disclosed by Xiang et al., U.S. Pat. No.7,563,821 and illustrated in Scheme 1.

Starting materials useful for preparing these compounds andintermediates thereof are commercially available or can be prepared bywell-known synthetic methods. Methods of synthesizing carboxyl esterlevodopa prodrugs are also described in Xiang et al., U.S. Pat. No.7,323,585, U.S. Pat. No. 7,342,131, U.S. Pat. No. 7,534,813, U.S. PatentApplication Publication No. 2008/0103200, U.S. Patent ApplicationPublication No. 2008/0171789, and U.S. Patent Application PublicationNo. 2008/0214663, each of which is incorporated by reference in itsentirety. Other methods for synthesizing anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate will be readilyapparent to one skilled in the art. Accordingly, the method presented inScheme 1 is illustrative rather than comprehensive.

For example, anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 can beprepared from the corresponding appropriately protected(2R)-2-phenylcarboxyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate precursor 2 via a director an indirect route as shown in Scheme 1.

When Pg is Boc (tert-butoxycarbonyl), treatment of precursor 2 with anappropriate acid such as hydrochloric acid in an organic solvent inwhich precursor 2 is soluble such as, for example, dioxane,dichloromethane, tetrahydrofuran, or mixtures of any of the foregoing atroom temperature, followed by solvent removal and crystallization of theresulting residue using an appropriate solvent such as acetonitrile, canprovide the hydrochloride salt 3. Other appropriate acids includevolatile acids such as trifluoroacetic acid and hydrogen bromide.Conversion of the hydrochloride salt 3 to the corresponding anhydrousmesylate salt 1 can be accomplished by neutralizing the hydrochloridesalt with an appropriate base such as sodium bicarbonate (NaHCO₃) orpotassium bicarbonate (KHCO₃) in an appropriate solvent such aswater/dichloromethane (DCM), separating DCM from the water, and addingmethanesulfonic acid to the DCM solution. The anhydrous mesylate salt 1can precipitate from the DCM solution.

In certain embodiments, precursor 2 can be directly converted to theanhydrous mesylate salt 1 by treating precursor 2 with an excess ofmethanesulfonic acid, e.g., about 1.1-100 equivalents, in an organicsolvent in which precursor 2 is soluble such as dioxane,dichloromethane, ethylacetate, methyl tert-butyl ether, tetrahydrofuran,or mixtures of any of the foregoing at a temperature from about 20° C.to about 100° C. The anhydrous mesylate salt 1 can then be precipitatedout in a non-polar solvent such as methyl tert-butyl ether (MTBE),dichloromethane, or mixtures of the foregoing.

In certain embodiments, precursor 2 can be converted to anhydrousmesylate salt 1 using a one-pot procedure by treating precursor 2 withan excess of hydrogen chloride in dioxane to produce the deprotectedhydrochloride salt 3, and then adding methanesulfonic acid to convertthe hydrochloride salt 3 to the anhydrous mesylate salt 1.

Anhydrous mesylate salt 1 can be crystallized from a solvent in whichthe mesylate salt 1 is soluble and in which the solubility of anhydrousmesylate salt 1 is temperature dependent, such as isopropanol,methanol/MTBE, 1% water in isopropanol, 1% water in acetonitrile, or 3%water in ethylacetate, to provide crystalline anhydrous mesylate salt 1.In certain embodiments, the solvent used for crystallizing anhydrousmesylate salt 1 can be selected from acetonitrile, methanol, ethanol,isopropanol, MTBE, dioxane, acetone, ethylacetate, ethylformate, hexane,dichloromethane, and mixtures of any of the foregoing. In certainsolvent mixtures comprising two solvents, the ratio of the two solventscan range from about 1:10 to about 10:1. In certain embodiments, thesolvent can further comprise less than about 10% water by volume, and incertain embodiments, less than about 5% water by volume. In certainembodiments, the solvent used for crystallizing anhydrous mesylate salt1 can comprise a mixture of methanol and MTBE in which the ratio (v/v)of methanol to MTBE is from about 1:5 to about 1:7. In certainembodiments, the solvent used for crystallizing anhydrous mesylate salt1 can comprise from about 1% to about 4% by volume water in isopropanol.

To prepare crystalline anhydrous mesylate salt 1, a solvent in which thesolubility of anhydrous mesylate salt 1 is temperature dependent andanhydrous mesylate salt 1, can be heated to provide a solution. Incertain embodiments, the solvent can be heated to a temperature up tothe reflux temperature, and in certain embodiments, up to a temperatureless than about 75° C. In certain embodiments, the concentration ofanhydrous mesylate salt 1 in the solution is less than about 500 mg/mLand in certain embodiments ranges from about 50 mg/mL to about 200mg/mL. The temperature of the solution can then be changed to decreasethe solubility of anhydrous mesylate salt 1 in the solvent. For example,the temperature of the solution can be decreased to room temperature(e.g., about 25° C.), and in certain embodiments to about 0° C. The timeto cool the solution can be selected to optimize the yield,compositional purity, and/or optical purity of crystalline anhydrousmesylate salt 1. In some embodiments, the solution can be cooled to afirst temperature and crystalline anhydrous mesylate salt 1 isolated,and the solution cooled further in a second crystallization andadditional crystalline anhydrous mesylate salt 1 isolated. Crystallineanhydrous mesylate salt 1 can be isolated from the solvent byfiltration. The filter cake can be washed in an appropriate solvent,such as, for example, a low boiling point solvent that minimizes theamount of residue remaining in crystalline anhydrous mesylate salt 1.Examples of appropriate wash solvents include acetonitrile, methanol,ethanol, isopropanol, MTBE, dioxane, acetone, ethylacetate,ethylformate, hexane, dichloromethane, and mixtures of any of theforegoing. One skilled in the art can appreciate that other methods canbe used to crystallize anhydrous mesylate salt 1, including, forexample, methods comprising stirring and/or seeding.

In certain embodiments, crystalline anhydrous mesylate salt 1 obtainedvia any of the methods disclosed herein is characterized by an X-raypowder diffraction having characteristic scattering angles measuredusing Cu—K_(α) radiation (° 2θ) at least at 4.7°±0.2°, 5.0°±0.2°,8.5°±0.2°, 9.6°±0.2°, 13.6°±0.2°, 15.0°±0.2°, 17.0°±0.2°, 17.4°±0.2°,17.7°±0.2°, 19.1°±0.2°, 19.5°±0.2°, 20.0°±0.2°, 20.4°±0.2°, 21.1°±0.2°,22.3°±0.2°, 22.9°±0.2°, 23.1°±0.2°, 23.3°±0.2°, 24.3°±0.2°, 25.0°±0.2°,25.3°±0.2°, 25.7°±0.2°, 25.8°±0.2°, 26.9°±0.2°, 27.3°±0.2°, 28.2°±0.2°,30.1°±0.2°, 30.5°±0.2°, 32.0°±0.2°, 33.8°±0.2°, 34.3°±0.2°, 37.6°±0.2°,and 38.4°±0.2°. In certain embodiments, crystalline anhydrous mesylatesalt 1 obtained via any of the methods disclosed herein is characterizedby an X-ray powder diffraction pattern having characteristic scatteringangles measured using Cu—K_(α) radiation (° 2θ) at least at 5.0°±0.2°,8.5°±0.2°, 13.6°±0.2°, 15.0°±0.2°, 17.0°±0.2°, 17.7°±0.2°, 20.4°±0.2°,21.1°±0.2°, 25.0°±0.2°, 25.8°±0.2°, 28.2°±0.2°, 30.1°±0.2°, and37.6°±0.2°.

In certain embodiments, crystalline anhydrous levodopa mesylate ischaracterized by an X-ray powder diffraction pattern havingcharacteristic scattering angles measured using Cu—K_(α) radiation (°2θ) at least at 5.0°±0.2°, 8.6°±0.2°, 9.6°±0.2°, 13.6°±0.2° 17.1°±0.2°,20.4°±0.2°, 25.7°±0.2°, and 28.3°±0.2°. In certain embodiments,crystalline anhydrous levodopa mesylate is characterized by an X-raypowder diffraction pattern having characteristic scattering anglesmeasured using Cu—K_(α) radiation (° 2θ) at least at 5.0°±0.2°,8.6°±0.2°, 9.6°±0.2°, 13.6°±0.2°, 17.1°±0.2°, 17.5°±0.2°, 17.7°±0.2°,19.2°±0.2°, 19.5°±0.2°, 20.4°±0.2°, 21.2°±0.2°, 25.1°±0.2°, 25.7°±0.2°,26.0°±0.2°, 28.3°±0.2°, 33.9°±0.2°, 37.7°±0.2°, and 38.4°±0.2°. Incertain embodiments, crystalline anhydrous levodopa mesylate ischaracterized by an X-ray powder diffraction pattern havingcharacteristic scattering angles measured using Cu—K_(α) radiation (°2θ) at least at 5.0°±0.2°, 8.6°±0.2°, 9.6°±0.2°, 10.2°±0.2°, 13.6°±0.2°,15.0°±0.2°, 17.1°±0.2°, 17.5°±0.2°, 17.7°±0.2°, 19.2°±0.2°, 19.5°±0.2°,20.4°±0.2°, 21.2°±0.2°, 25.1°±0.2°, 25.3°±0.2°, 25.7°±0.2°, 26.0°±0.2°,26.9°±0.2°, 27.3°±0.2°, 28.3°±0.2°, 30.5° 10.2°, 33.9°±0.2°, 34.3°±0.2°,35.2°±0.2°, 37.7°±0.2°, and 38.4°±0.2°.

In certain embodiments, formation and crystallization of anhydrousmesylate salt 1 can be performed in a one-pot procedure at about roomtemperature, e.g., 25° C. For example, after deprotection andneutralization, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate can be dissolved in asolvent such as ethylacetate, isopropanol/dichloromethane, orisopropanol/ethylacetate and treated with about 0.9-1.2 equivalents ofmethanesulfonic acid at about room temperature. Anhydrous mesylate salt1 can crystallize from the solution with or without stirring or seeding.

As an example of a one-pot procedure for preparing crystalline anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, a solution of(2R)-2-phenylcarbonyloxypropyl(2S)-2-(tert-butoxycarbonyl)amino-3-(3,4-dihydroxyphenyl)propanoate in afirst solvent in which it is soluble is prepared. Examples of suitablesolvents include dichloromethane and dioxane. The tert-butoxycarbonylgroup is deprotected by adding an acid to provide(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate acid salt. Suitable acidsare not limited to volatile acids. Examples of suitable acids fordeprotecting the tert-butoxycarbonyl group include hydrochloric acid,methanesulfonic acid, trifluoroacetic acid, and hydrogen bromide. Afterdeprotection, the first solvent can be removed and water added to the(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate acid salt. The(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate acid salt can beneutralized with a base such as NaHCO₃ or KHCO₃ to provide(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate.(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate can then be extractedwith a second solvent such as methyl tert-butylether, dichloromethane,ethylacetate, or a mixture of ethylacetate and isopropanol.Methanesulfonic acid can be added to the extracted(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate to convert the(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate to crystalline anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate. Crystallineanhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate can then beisolated from the second solvent by filtration.

One skilled in the art will appreciate that the methods provided by thepresent disclosure can be used to prepare anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 or crystallineform thereof having high compositional and diastereomeric purity. Forexample, in certain embodiments, the compositional purity of anhydrouslevodopa mesylate salt 1 can be at least about 95%, in certainembodiments, at least about 97%, in certain embodiments, at least about98%, and in certain embodiments, can be at least about 99%, and incertain embodiments, the diastereomeric purity can be at least about95%, in certain embodiments, at least about 97%, in certain embodiments,at least about 98%, and in certain embodiments, at least about 99%.

Crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isformed from crystalline anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate as a result ofthermodynamic and kinetic equilibria of the two crystal forms with waterin the environment.

Crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beformed from a solvent containing anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and having awater activity greater than about 0.6, and in certain embodiments,greater than about 0.7, wherein the water activity is determined at roomtemperature (about 25° C.). In certain embodiments, the solvent may be amixture of an alcohol and water, and in certain embodiments, a mixtureof isopropanol and water. In certain embodiments, the solvent comprisesisopropanol and at least about 7%-bv water. At room temperature (about25° C.) a mixture of isopropanol and about 7 vol-% to about 10 vol-%water has a water activity ranging from about 0.6 to about 0.7.Crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beprepared from other solvents containing (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and at atemperature of about 25° C. providing the water activity of the mixtureis greater than about 0.6 or greater than about 0.7. At highertemperatures, the critical water activity will be higher.

X-ray powder diffraction patterns of crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate prepared fromisopropanol solutions at about 25° C. having different water content areshown in FIG. 5. Above a water content greater than about 10 vol-%,crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratecrystallizes from the solution, whereas at lower water content,crystalline anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate is formed.

Crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate mayalso be produced during high shear wet granulation processing ofcrystalline anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate. For example,crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate can beformed during high shear wet granulation processing of a mixturecontaining crystalline anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and an amount ofvehicle using a water content greater than about 10 wt-%, greater thanabout 12 wt-%, and in certain embodiments, greater than about 15 wt-%.In certain embodiments, the amount of water used during high shear wetgranulation processing can range from about 10 wt-% to about 20 wt-%,and in certain embodiments, about 15 wt-%. In certain embodiments, thewater content can be greater than about 20 wt-%.

The amount of vehicle used to form granules comprising(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate can beless than about 10 wt-%, less than about 5 wt-%, and in certainembodiments, less than about 1 wt-%. X-ray powder diffraction patternsof granules comprising a mixture of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateprepared by high shear wet granulation using a water content of about 15wt-% are shown in FIG. 4.

Crystalline anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate can also convertto crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate in thepresence of water vapor. For example, crystalline anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate converts to thehydrate within 24 hours at a temperature of about 25° C. and a humidityof at least about 60% RH.

One skilled in the art will appreciate that although(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isdisclosed, a sample of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate canhave various compositional and diastereomeric purities. In certainembodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orcrystalline form thereof can exhibit a compositional purity of at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, and in certainembodiments, in excess of at least about 99%. In certain embodiments,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orcrystalline form thereof can exhibit a diastereomeric purity of at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, and in certainembodiments, in excess of at least about 99%.

Furthermore, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate may comprise acombination of anhydrous and hydrated forms.

In certain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate is acrystalline form. In certain embodiments, crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateexhibits an X-ray powder diffraction pattern substantially as shown inany one of FIGS. 4, 5, 7, and 8.

In certain embodiments, crystalline levodopa mesylate, hydrate ischaracterized by an X-ray powder diffraction pattern havingcharacteristic scattering angles measured using Cu—K_(α) radiation (°2θ) at least at 6.0°±0.2°, 9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°,14.6°±0.2°, 15.1°±0.2°, 15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°,18.5°±0.2°, and 19.2°±0.2°.

In certain embodiments, crystalline levodopa mesylate, hydrate ischaracterized by an X-ray powder diffraction pattern havingcharacteristic scattering angles measured using Cu—K_(α) radiation (°2θ) at least at 6.0°±0.2°, 9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°,14.6°±0.2°, 15.1°±0.2°, 15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°,18.5°±0.2°, 19.2°±0.2°, 20.8°±0.2°, 21.9°±0.2°, 22.8°±0.2°, 23.4°±0.2°,23.7°±0.2°, 23.9°±0.2°, and 26.5°±0.2°.

In certain embodiments, crystalline levodopa mesylate, hydrate ischaracterized by an X-ray powder diffraction pattern havingcharacteristic scattering angles measured using Cu—K_(α) radiation (°2θ) at least at 6.0°±0.2°, 9.1°±0.2°, 9.6°±0.2°, 11.2°±0.2°, 12.0°±0.2°,12.8°±0.2°, 13.8°±0.2°, 14.3°±0.2°, 14.6°±0.2°, 15.1°±0.2°, 15.6°±0.2°,16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°, 18.5°±0.2°, 18.7°±0.2°, 19.2°±0.2°,20.5°±0.2°, 20.8°±0.2°, 21.1°±0.2°, 21.9°±0.2°, 22.8°±0.2°, 23.4°±0.2°,23.7°±0.2°, 23.9°±0.2°, 24.7°±0.2°, 26.5°±0.2°, 28.2°±28.3°±0.2°, and29.5°±0.2°.

One skilled in the art will recognize that slight variations in theobserved ° 2θ diffraction angles can be expected based on, for example,the specific diffractometer employed, the analyst, and the samplepreparation technique. Greater variation can be expected for therelative peak intensities. Comparison of diffraction patterns can bebased primarily on observed °2θ diffraction angles with lesserimportance attributed to relative peak intensities.

In certain embodiments, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate ischaracterized by a differential scanning calorimetry (DSC) thermogramhaving an endotherm at about 75° C. representing dehydration followed byan exotherm at about 80° C. representing recrystallization to theanhydrous form. The anhydrous form then exhibits an endotherm at about161° C. An example of a DSC thermogram of crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isshown in FIG. 2.

In certain embodiments, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratecomprises from about 1.2 moles water to about 1.8 moles water, fromabout 1.4 moles water to about 1.6 moles water, and in certainembodiments, about 1.5 moles water per mole of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate.

In certain embodiments, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratecomprises from about 5.1 wt-% water to about 5.9 wt-% water, from about5.4 wt-% water to about 5.6 wt-% water, and in certain embodiments,about 5.5 wt-% water.

In certain embodiments, crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isstable, e.g., does not absorb moisture and/or convert to anotherpolymorphic form under typical pharmaceutical processing and/or storageconditions.

The physical properties and characteristics of crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateprepared by methods provided by the present disclosure are consistentwith that of a single polymorph.

Pharmaceutical compositions provided by the present disclosure maycomprise a therapeutically effective amount of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate, and incertain embodiments, in purified form, together with a suitable amountof one or more pharmaceutically acceptable vehicles, so as to provide acomposition for proper administration to a patient. Other examples ofsuitable pharmaceutical vehicles are described in the art.

Pharmaceutical compositions comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablevehicles, which facilitate processing of levodopa prodrug mesylate orcrystalline form thereof and one or more pharmaceutically acceptablevehicles into formulations that can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.Pharmaceutical compositions provided by the present disclosure may takethe form of solutions, suspensions, emulsion, tablets, pills, pellets,capsules, capsules containing liquids, powders, sustained-releaseformulations, suppositories, emulsions, aerosols, sprays, suspensions,or any other form suitable for use.

In certain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beincorporated into pharmaceutical compositions to be administered orally.Oral administration of such pharmaceutical compositions may result inuptake of the (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratethroughout the intestine and entry into the systemic circulation. Suchcompositions may be prepared in a manner known in the pharmaceutical artand comprise (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate and atleast one pharmaceutically acceptable vehicle. Pharmaceuticalcompositions may include a therapeutically effective amount of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate, insome embodiments, in purified form, together with a decarboxylaseinhibitor such as carbidopa or benserazide, and/or a COMT inhibitor suchas entacapone and tolecapone, and a suitable amount of apharmaceutically acceptable vehicle.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beadministered to a patient in a therapeutically effective amount. It willbe understood, however, that the amount of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateactually administered will be determined by a physician, in the light ofthe relevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the disease beingtreated, the severity of the patient's symptoms, and the like.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beused with a number of different dosage forms, which can be adapted toprovide sustained release of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate uponoral administration. Sustained release refers to release of atherapeutic or preventive amount of a drug or an active metabolitethereof over a period of time that is longer than that of a conventionalformulation of the drug. For oral formulations, sustained releasetypically means release of the drug within the gastrointestinal tractlumen over a time period ranging, for example, from about 2 to about 30hours, and in certain embodiments, over a time period ranging from about4 to about 24 hours. Sustained release formulations achievetherapeutically effective concentrations of the drug in the systemiccirculation over a prolonged period of time relative to that achieved byoral administration of a conventional formulation of the drug. Sustainedrelease oral dosage forms are known in the art.

Regardless of the specific form of sustained release oral dosage formused, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may bereleased from a dosage form such as an orally administered dosage form,over a sufficient period of time to provide prolonged therapeuticconcentrations of levodopa in the blood of a patient enablingadministration of the dosage form on only a once or twice per day basis.Following oral administration, dosage forms comprising(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate canprovide a therapeutic or prophylactic concentration of levodopa in theplasma and/or blood of a patient for a time period of at least about 4hours, in certain embodiments, for at least about 8 hours, for at leastabout 12 hours, for at least about 16 hours, for at least about 20hours, and in certain embodiments, for at least about 24 hours followingoral administration of the dosage form to the patient. A therapeuticallyor prophylactically effective concentration of levodopa in the bloodand/or plasma of a patient can depend on a number of factors including,for example, the disease being treated, the severity of the disease, theweight of the patient, and the health of the patient.

In certain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orpharmaceutical compositions thereof may be administered twice per day,in certain embodiments, once per day, and in certain embodiments atintervals greater than once per day.

In certain embodiments, an oral dosage form comprises(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate; and incertain embodiments, a combination of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate andanhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate. In certainembodiments, the amount of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate in theoral dosage form is at least about 75 wt-%, at least about 80 wt-%, atleast about 85 wt-%, and in certain embodiments, at least about 90 wt-%.In certain embodiments, the combined amount of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate andanhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate in the oraldosage form is at least about 75 wt-%, at least about 80 wt-%, at leastabout 85 wt-%, and in certain embodiments, at least about 90 wt-%.

Pharmaceutical compositions provided by the present disclosure may beadministered for therapeutic or prophylactic treatments. A therapeuticamount is an amount sufficient to treat a disease or symptom of adisease, or otherwise prevent, hinder, retard, or reverse theprogression of disease or undesirable symptom of a disease. Inprophylactic applications, pharmaceutical compositions provided by thepresent disclosure may be administered to a patient susceptible to orotherwise at risk of a particular disease or infection. Hence, aprophylactically effective amount is an amount sufficient to prevent,hinder or retard a disease state or its symptoms.

An appropriate dosage of the pharmaceutical composition may bedetermined according to any one of several well-established protocols.For example, animal studies, such as studies using mice or rats, may beused to determine an appropriate dose of a pharmaceutical compound. Theresults from animal studies can be extrapolated to determine doses foruse in other species, such as for example, humans. For example, theefficacy of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate andcompositions thereof for treating Parkinson's disease may be assessedusing animal models of Parkinson's disease and in clinical studies.Animal models of Parkinson's disease are known (O'Neil et al., CNS DrugRev. 2005, 11(1), 77-96; Faulkner et al., Ann. Pharmacother. 2003,37(2), 282-6; Van Blercom et al., Clin Neuropharmacol. 2004, 27(3),124-8; Cho et al., Biochem. Biophys. Res. Commun. 2006, 341, 6-12; andEmborg, J. Neuro. Meth. 2004, 139, 121-143).

Levodopa prodrugs are precursors of dopamine. Thus, levodopa prodrugmesylate, hydrate provided by the present disclosure may be administeredto a patient suffering from any disease or disorder for which the parentdrug, levodopa, is known or hereafter discovered to be therapeuticallyeffective. Levodopa prodrug mesylate, hydrate may be administered to apatient, such as a human, to treat a disease or disorder such asParkinson's disease. The methods comprise administering to a patient inneed of such treatment a therapeutically effective amount of levodopaprodrug mesylate, hydrate. In therapeutic methods provided by thepresent disclosure, a therapeutically effective amount of levodopaprodrug mesylate, hydrate may be administered to a patient sufferingfrom a disease such as Parkinson's disease, depression, attentiondeficit disorder, schizophrenia, manic depression, cognitive impairmentdisorders, restless legs syndrome, periodic limb movement disorders,tardive dyskinesia, Huntington's disease, Tourette's syndrome,hypertension, addictive disorders, congestive heart failure, orexcessive daytime sleepiness. In prophylactic methods provided by thepresent disclosure a therapeutically effective amount of levodopaprodrug mesylate, hydrate may be administered to a patient at risk ofdeveloping a disease such as Parkinson's disease, depression, attentiondeficit disorder, schizophrenia, manic depression, cognitive impairmentdisorders, restless legs syndrome, periodic limb movement disorders,tardive dyskinesia, Huntington's disease, Tourette's syndrome,hypertension, addictive disorders, congestive heart failure, orexcessive daytime sleepiness.

Levodopa is well known to be effective in treating Parkinson's disease(see Fahn et al., N Engl J Med 2004, 351(24), 2498-2508).

Low doses of levodopa given as adjunctive treatment with typicalantipsychotic drugs improves the clinical outcome in schizophrenia (seeJaskiw and Popli, Psychopharmacology 2004, 171, 365-374) and suggests anenhanced effect on negative symptoms and cognitive impairment withoutworsening of psychotic symptoms (Alpert and Friedhoff, Am J Psychiatry1980, 135, 1329-32; Bruno and Bruno, Acta Psychiatr Scand, 1966, 42,264-71; Buchanan et al., Aust N Z J Psychiatry 1975, 9, 269-71; Gerlachand Luhdorf, Psychopharmacologia 1975, 44, 105-110; Inanaga et al.,Folia Psychiatr Neurol Jpn 1975, 29, 123-43; and Kay and Opler, Int JPsychiat Med 1985-86, 15, 293-98).

Levodopa has been shown to be effective in treating cognitive impairmentdisorders such as cognitive dysfunction in patients with Parkinson'sdisease (Kulisevsky, Drugs Aging 2000, 16(5), 365-79) and improving wordlearning in healthy patients (Knecht et al., Ann. Neurol 2004, 56(1),20-6).

Levodopa has been shown to be effective in treating restless legssyndrome (Ondo and Jankovic, Neurology 1996, 47, 14354; and von Scheele,Lancet 1986, 2(8504), 426-7).

Studies suggest that levodopa can be useful in treating periodicmovement disorders such as tardive dyskinesia (Rascol and Fabre,Clinical Neuropharmacology 2001, 24(6), 313-323; Soares and McGrath,Schizophr Res 1999, 39(1), 1-16; and Ebadi and Srnivasan,Pharmacological Reviews 1996, 47(4), 575-604), dystonia (Jankovic,Lancet Neurol 2006, 5, 864-72), and motor recovery after stroke(Scheidtmann et al., The Lancet, 2001, 358, 787-790; and Floel et al.,Neurology 2005, 65(3), 472-4).

Tardive dyskinesia is a type of dyskinesia that is distinct fromlevodopa-induced dyskinesia (Rascol and Fabre, ClinicalNeuropharmacology 2001, 24(6), 313-323). Levodopa-induced dyskinesia isusually associated with the treatment of Parkinson's disease. Tardivedyskinesia is a type of dyskinesia that is typically induced byneuroleptics, i.e. drugs used for treating psychotic disorders. Forexample, Soares and McGrath, Schizophr Res 1999, 39(1), 1-16 disclosethat levodopa can be used to treat tardive dyskinesia.

Levodopa has shown effectiveness in treating rigidity associated withHuntington's disease (Racette and Perlmutter, J Neurol NeurosurgPsychiatry 1998, 65(4), 577-9).

The use of levodopa for treating hypertension is disclosed, for example,by Doggrell, Expert Opin Investig Drugs 2002, 11(5), 631-44. Low doselevodopa can be used to induce diuresis and naturesis, which can beuseful in treating hypertension.

The use of levodopa for treating excessive daytime sleepiness,especially excessive daytime sleepiness associated with Parkinson'sdisease is disclosed, for example, by O'Sulleabhaim and Dewey, ArchNeurol 2002, 59(6), 986-989. Levodopa has also been shown to beeffective in treating excessive daytime sleepiness in patients withnarcolepsy (Boivin and Montplaisir, Neurology 1991, 41, 1267-1269) andhypersomnia (Silber, Neurologic Clinics 2001, 19(1), 173-86; Paus etal., Movement Disorders 2003, 18(6), 659-667; Hogl et al., MovementDisorders 2003, 18(3), 319-323; and O'Suilleabhain and Dewey, ArchNeurol 2002, 59, 986-989).

In certain embodiments, levodopa prodrug mesylate, hydrate orpharmaceutical composition thereof may be co-administered with anothertherapeutic agent or drug, such as a decarboxylase inhibitor such ascarbidopa or benserazide, which may act as a protectant to inhibit orprevent premature decarboxylation of the levodopa prodrug mesylate,hydrate and/or the levodopa metabolite. Levodopa prodrug mesylatehydrate may be delivered from the same dosage form as the L-aromaticamino acid decarboxylase inhibitor or from a different dosage form.Levodopa prodrug mesylate, hydrate may be administered at the same timeas, prior to, or subsequent to, the administration of a decarboxylaseinhibitor. Levodopa prodrug mesylate, hydrate together with adecarboxylase inhibitor can be administered to a patient, such as ahuman, to treat a disease such as Parkinson's disease.

In certain embodiments, levodopa prodrug mesylate or pharmaceuticalcomposition thereof together with at least one decarboxylase inhibitormay be used to treat a disease for which levodopa is known or believedto be effective in treating. In certain embodiments, levodopa prodrugmesylate, hydrate or pharmaceutical composition thereof may be usefulfor the treatment of Parkinson's disease. When used to treat Parkinson'sdisease, levodopa prodrug mesylate, hydrate or pharmaceuticalcomposition thereof may be administered or applied in combination with adecarboxylase inhibitor such as carbidopa and/or benserazide.Additionally, the therapeutic effectiveness of the above combinationsmay be enhanced by co-administration of another pharmaceutically activeagent such as a catechol-O-methyltransferase (COMT) inhibitor such asentacapone and/or tolecapone. Further, in certain embodiments, levodopaprodrug mesylate, hydrate or pharmaceutical composition thereof may beadministered to a patient, such as a human, together with (i) adecarboxylase inhibitor such as carbidopa or benserazide, (ii) a COMTinhibitor such as entacopone or tolecapone, or (iii) a combination of adecarboxylase inhibitor such as carbidopa or benserazide and a COMTinhibitor such as entacopone or tolecapone, to treat a disease ordisorder such as Parkinson's disease.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beincluded in a pharmaceutical composition and/or dosage form adapted fororal administration, although (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate mayalso be administered by any other convenient mute, such as for example,by injection, infusion, inhalation, transdermal, or absorption throughepithelial or mucosal membranes (e.g., oral, rectal, and/or intestinalmucosa).

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orpharmaceutical compositions thereof may provide therapeutic orprophylactic plasma and/or blood concentrations of levodopa followingoral administration to a patient. The carboxyl ester promoiety of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may becleaved in vivo either chemically and/or enzymatically to release theparent drug, levodopa. One or more enzymes present in the stomach,intestinal lumen, intestinal tissue, blood, liver, brain, or any othersuitable tissue of a patient may enzymatically cleave the promoiety ofthe administered compounds. For example, the carboxyl ester promoiety of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may becleaved prior to absorption from the gastrointestinal tract (e.g.,within the stomach or intestinal lumen) and/or after absorption from thegastrointestinal tract (e.g., in intestinal tissue, blood, liver, orother suitable tissue of a mammal). In certain embodiments,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beactively transported across the intestinal endothelium by organic cationtransporters expressed throughout the gastrointestinal tract includingthe small intestine and colon. Levodopa may remain conjugated to thecarboxyl ester promoiety during transit across the intestinal mucosalbarrier to prevent or minimize presystemic metabolism. In certainembodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isessentially not metabolized to levodopa within gastrointestinalenterocytes, but is metabolized to levodopa within the systemiccirculation, for example in the plasma. In such embodiments,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate may be absorbedinto the systemic circulation from the small and large intestines eitherby active transport, passive diffusion, or by both active and passiveprocesses. Cleavage of the promoiety from (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate afterabsorption from the gastrointestinal tract may allow the levodopaprodrug mesylate, hydrate to be absorbed into the systemic circulationeither by active transport, passive diffusion, or by both active andpassive processes. The mechanism of cleavage is not important to thepresent embodiments. For example, the carboxyl ester promoiety can becleaved after absorption from the gastrointestinal tract, for example,in intestinal tissue, blood, liver, or other suitable tissue of amammal.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beadministered to achieve therapeutically effective levodopa plasmaconcentrations and using a similar dosing regimen as described in theart for levodopa. For example, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate can beuseful in treating Parkinson's disease by administration of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratetogether with a decarboxylase inhibitor such as carbidopa, in certainembodiments by the oral route, to a mammalian subject in need of thetreatment. In a human subject weighing about 70 kg,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate can beadministered at a dose over time having an equivalent weight of levodopafrom about 10 mg to about 10 g per day, and in certain embodiments, anequivalent weight of levodopa from about 100 mg to about 3 g per day.Accordingly, a dose of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate can beadministered at a dose over time from about 20 mg to about 20 g per day,and in certain embodiments, from about 200 mg to about 7 g per day. Adose of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate takenat any one time can have an equivalent weight of levodopa from about 10mg to about 3 g, and in certain embodiments, from about 100 mg to about2 g. A dose of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate takenat any one time can be from about 20 mg to about 7 g, and in certainembodiments, from about 200 mg to about 5 g. A dose can be adjusted byone skilled in the art based on several factors, including, for example,the body weight and/or condition of the subject treated, the dose of thedecarboxylase inhibitor being administered, the severity of the diseasebeing treated, the incidence of side effects, the manner ofadministration, and the judgment of the prescribing physician.Appropriate doses and regimens may be determined by methods known to oneskilled in the art.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beassayed in vitro and in vivo for the desired therapeutic or prophylacticactivity prior to use in humans. In vivo assays may also be used todetermine whether administration of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate istherapeutically effective. (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate mayalso be demonstrated to be effective and safe using animal modelsystems.

In certain embodiments, a therapeutically effective dose of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate mayprovide therapeutic benefit without causing substantial toxicity.Toxicity of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may bedetermined using standard pharmaceutical procedures and may beascertained by one skilled in the art. The dose ratio between toxic andtherapeutic effect is the therapeutic index. A dosage of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may bewithin a range capable of establishing and maintaining a therapeuticallyeffective circulating plasma and/or blood concentration of levodopa thatexhibits little or no toxicity.

In addition to the use of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate andcompositions comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateprovided by the present disclosure for treating Parkinson's disease,levodopa prodrugs mesylate, hydrate and compositions thereof may also beuseful for treating other dopamine-related diseases. Dopamine-relateddiseases can be characterized by either insufficient or excessivefunctional dopaminergic activity in the central nervous system. Examplesof other dopamine-related diseases include, but are not limited to,affective disorders such as depression and attention deficit disorder;psychotic disorders such as schizophrenia and manic depression;cognitive impairment disorders such as mild cognitive impairment;movement disorders such as restless legs syndrome, periodic limbmovement disorders, tardive dyskinesia, hypertension, Huntington'sdisease, and Tourette's syndrome; addictive disorders such as alcoholaddiction or abuse, nicotine addiction or abuse, and drug addiction andabuse; congestive heart failure; and excessive daytime sleepiness. Forthe treatment of these and other dopamine-related diseases,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beco-administered with an additional active agent such as, for example, adecarboxylase inhibitor and/or a COMT inhibitor. Therapeuticallyeffective doses for treating dopamine-related diseases may be determinedby methods disclosed herein for the treatment of Parkinson's diseaseand/or by methods known in the art. (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orpharmaceutical compositions thereof may also be used to modulatedopamine levels in a patient.

In certain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beused in combination therapy with at least one other therapeutic agent.Pharmaceutical compositions provided by the present disclosure mayinclude, in addition to (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate one ormore therapeutic agents effective for treating the same or differentdisease, disorder, or condition.

Methods provided by the present disclosure include administration of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orpharmaceutical compositions thereof and one or more other therapeuticagents, provided that the combined administration does not inhibit thetherapeutic efficacy of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orlevodopa and/or does not produce adverse combination effects.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate andanother therapeutic agent or agents may act additively orsynergistically. In certain embodiments, pharmaceutical compositionsprovided by the present disclosure can be administered concurrently withthe administration of another therapeutic agent, which may be containedin the same pharmaceutical composition as, or in a different compositionfrom that containing (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate. Incertain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beadministered prior or subsequent to administration of anothertherapeutic agent. In certain embodiments of combination therapy, thecombination therapy can comprise alternating between administering acomposition provided by the present disclosure and a compositioncomprising another therapeutic agent, e.g., to minimize adverse sideeffects associated with a particular drug. When(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isadministered concurrently with another therapeutic agent that canpotentially produce adverse side effects including, but not limited to,toxicity, the therapeutic agent may advantageously be administered at adose that falls below the threshold at which the adverse side effect iselicited.

In certain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate mayfurther be administered together with one or more compounds thatenhance, modulate, and/or control the release, bioavailability,therapeutic efficacy, therapeutic potency, and/or stability of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate and/orlevodopa. For example, to enhance therapeutic efficacy levodopa prodrugmesylate hydrate may be co-administered with one or more active agentsto increase the absorption or diffusion of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate and/orlevodopa through the gastrointestinal tract, or to modify degradation ofthe (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate and/orlevodopa in the systemic circulation. In certain embodiments,(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beco-administered with an active agent having pharmacological effects thatenhance the therapeutic efficacy of levodopa after being released from(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate. Incertain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beco-administered with an active agent having pharmacological effects thatenhance the therapeutic efficacy of dopamine after being released fromlevodopa.

In certain embodiments, (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate orpharmaceutical compositions comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate may beadministered to a patient together with another compound for treatingParkinson's disease, depression, attention deficit disorder,schizophrenia, manic depression, cognitive impairment disorders,restless legs syndrome, periodic limb movement disorders, tardivedyskinesia, Huntington's disease, Tourette's syndrome, hypertension,addictive disorders, congestive heart failure, or excessive daytimesleepiness.

Examples of drugs useful for treating Parkinson's disease includeamantadine, baclofen, biperiden, benztropine, orphenadrine,procyclidine, trihexyphenidyl, levodopa, carbidopa, andropinirole,apomorphine, benserazide, bromocriptine, budipine, cabergoline,eliprodil, eptastigmine, ergoline, galanthamine, lazabemide, lisuride,mazindol, memantine, mofegiline, pergolide, piribedil, pramipexole,propentofylline, rasagiline, remacemide, ropinirole, selegiline,spheramine, terguride, entacapone, and tolcapone.

Examples of drugs useful for treating mood disorders such as depressioninclude tricyclic antidepressants such as amitriptyline, amoxapine,clomipramine, desipramine, doxepin, imipramine, maprotiline,nortriptyline, protriptyline, and trimipramine; selective serotoninreuptake inhibitors such as citalopram, escitalopram, fluoxetine,fluvoxamine, paroxetine, and sertraline; serotonin-noradrenalinereuptake inhibitors such as venlafaxine, duloxetine, sibutramine, andmilnacipran; monoamine oxidase inhibitors such as phenelzine andtranylcypromine; and psychostimulants such as dextroamphetamine andmethylphenidate. Other antidepressants include benmoxine, butriptyline,dosulepin, imipramine, kitanserin, lofepramine, medifoxamine, mianserin,mirtazapine, viloxazine, cotinine, nisoxetine, reboxetine, tianeptine,acetaphenazine, binedaline, brofaromine, cericlamine, clovoxamine,iproniazid, isocarboxazid, moclobemide, phenyhydrazine, selegiline,sibutramine, ademetionine, adrafinil, amesergide, amisulpride,amperozide, benactyzine, bupropion, caroxazone, gepirone, idazoxan,metralindole, minaprine, nefazodone, nomifensine, ritanserin, roxindole,S-adenosylmethionine, escitalopram, tofenacin, trazodone, tryptophan,zalospirone, and Saint John's wort.(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoatemesylate, hydrate and pharmaceutical compositions thereof may also beused in conjunction with psychotherapy or electroconvulsive therapy totreat mood disorders such as depression.

Examples of drugs useful for treating attention deficit disorder includeatomoxetine, bupropion, dexmethylphenidate, dextroamphetamine,metamphetamine, methylphenidate, and pemoline.

Examples of drugs for treating schizophrenia include aripiprazole,loxapine, mesoridazine, quetiapine, reserpine, thioridazine,trifluoperazine, and ziprasidone. The use of levodopa prodrugs incombination with at least one antipsychotic agent in an amount that iseffective for treating a positive symptom of schizophrenia is disclosedin Tran, U.S. Patent Application Publication No. 2008/0070984, which isincorporated by reference herein in its entirety. The at least oneantipsychotic agent can be chosen from, for example, chlorpromazine,haloperidol, fluphenazine, loxapine, mesoridazine, molindone,perphenazine, pimozide, raclopride, remoxipride, thioridazine,thiothixene, and trifluoperazine.

Examples of drugs useful for treating manic depression includecarbamazepine, clonazepam, clonidine, valproic acid, verapamil,lamotrigine, gabapentin, topiramate, lithium, clozapine, olanzapine,risperidone, quetiapine, ziprasidone, clonazepam, lorazepam, zolipidem,St. John's wort, and omega-3 fatty acids.

Examples of drugs useful for treating cognitive or memory disordersinclude antipsychotic drugs such as chlorpromazine, fluphenazine,haloperidol, loxapine, mesoridazine, molindone, perphenazine, pimozide,thioridazine, thiothixene, trifluoperazine, aripiprazole, clozapine,olanzapine, quetiapine, risperidone, and ziprasidone; sedatives such asdiazepam and lorazepam; benzodiazepines such as alprazolam,chlordiazepoxide, clonazepam, clorazepate, diazepam, lorazepam, andoxazepam; nonsteroidal anti-inflammatory drugs such as aceclofenac,acetaminophen, alminoprofen, amfenac, aminopropylon, amixetrine,aspirin, benoxaprofen, bromfenac, bufexamac, carprofen, celecoxib,choline, salicylate, cinchophen, cinmetacin, clopriac, clometacin,diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen,indomethacin, indoprofen, ketoprofen, ketorolac, mazipredone,meclofenamate, nabumetone, naproxen, parecoxib, piroxicam, pirprofen,rofecoxib, sulindac, tolfenamate, tolmetin, and valdecoxib;acetylcholinesterase inhibitors such as donepezil, galantamine,rivastigmine, physostigmine, and tacrine; and N-methyl-D-aspartate(NMDA) receptor blockers such as memantine.

Examples of drugs useful for treating restless legs syndrome includedopaminergics such as levodopa, pergolide mesylate, pramipexole, andrinirole hydrochloride, benzodiazepines such as clonazepam and diazepam,opioids such as codeine, propoxyphene, and oxycodone, andanticonvulsants such as gabapentin, prebabalin, and carbamazepine.

Examples of drugs useful for treating movement disorders such as tardivedyskinesia include reserpine, tetrabenazine, and vitamin E.

Examples of drugs useful for treating Huntington's disease includeantipsychotics such as haloperidol, chlorpromazine, and olanzapine;antidepressants such as fluoxetine, sertraline hydrochloride, andnortriptyline; tranquilizers such as benzodiazepines, paroxetine;venlafaxin, and beta-blockers; mood-stabilizers such as lithium,valproate, and carbamazepine; and Botulinum toxin.

Examples of drugs useful for treating Tourette's syndrome includehaloperidol, pergolide, and pimozide.

Examples of drugs useful for treating hypertension include acebutolol,amiloride, amlodipine, atenolol, benazepril, betaxolol, bisoprolol,candesartan captopril, careolol, carvedilol, chlorothiazide,chlorthalidone, clonidine, diltiazem, doxazosin, enalapril, eplerenone,eprosartan, felodipine, fosinopril, furosemide, guanabenz, guanethidine,guanfacine, hydralazine, hydrochlorothiazide, indapamide, irbesartan,isradipine, labetalol, lisinopril, losartan, methyldopa, metolazone,metoprolol, minoxidil, moexipril, nadolol, nicardipine, nifedipine,nisoldipine, nitroglycerin, olmesartan, perindopril, pindolol, prazosin,propranolol, quinapril, ramipril, reserpine, spironolactone,telmisartan, terazosin, timolol, torsemide, trandolapril, valsartan, andverapamil.

Examples of drugs useful for treating alcohol addiction or abuse includedisulfiram, naltrexone, clonidine, methadone, 1-α-acetylmethadol,buprenorphine, and bupropion.

Examples of drugs useful for treating narcotic addiction or abuseinclude buprenorphine, tramadol, methadone, and naltrexone.

Examples of drugs useful for treating nicotine addiction or abuseinclude bupropion, clonidine, and nicotine.

Examples of drugs useful for treating congestive heart failure includeallopurinol, amiloride, amlodipine, benazepril, bisoprolol, carvedilol,digoxin, enalapril, eplerenone, fosinopril, furosemide,hydrochlorothiazide, hydralazine, isosorbide dinitrate, isosorbidemononitrate, lisinopril, metoprolol, moexipril, nesiritide, nicardipine,nifedipine, nitroglycerin, perindopril, prazosin, quinapril, ramipril,spironolactone, torsemide, trandolapril, triamcinolone, and valsartan.

Examples of drugs useful for treating excessive daytime sleepinessinclude dextroamphetamine, methylphenidate, modafinil, and sodiumoxybate.

The following examples describe in detail preparation of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate,pharmaceutical compositions thereof, and uses thereof. It will beapparent to one skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from the scopeof the disclosure. Examples 11 and 12 are prophetic.

EXAMPLE 1 (2R)-2-Phenylcarbonyloxypropyl(2S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate, Anhydrous (1)Step A:(2S)-3-(3,4-Dihydroxyphenyl)-2-[(tert-butoxycarbonyl)amino]propanoicacid, tetrabutylammonium salt (1a)

A solution of N-Boc-(L)-Dopa (175 g, 0.59 mol) in methanol (1 L) wascautiously mixed with a methanolic solution of tetrabutylammoniumhydroxide (1.0 M, 0.55 L) at 0° C. for 30 min. The mixture was thenconcentrated under reduced pressure and dried by azeotroping twice withtoluene. The residue was crystallized after cooling at 4° C. for 16 h.The resulting crystalline solid was washed with acetone (400 mL×3),collected on a Buchner funnel, and then dried under high vacuum toafford 245 g (83% yield) of the title compound 1a. ¹H NMR (400 MHz,DMSO-d₆): δ 0.94 (t, J=7.6 Hz, 12H), 1.30 (m, 17H), 1.60 (m, 8H), 3.18(m, 8H), 4.58 (m, 1H), 5.68 (d, J=5.6 Hz, 1H), 6.30 (d, J=7.6 Hz, 1H),6.46 (d, J=8.0 Hz, 1H), 6.51 (s, 1H), 8.85 (s, 1H); 8.94 (s, 1H).

Step B: (1R)-2-Bromo-1-methylethyl benzoate (1b)

A solution of (2R)-propylene glycol (20.0 g, 262.8 mmol), benzaldehyde(33.4 mL, 328.6 mmol, 1.25 eq) and p-toluenesulfonic acid (2.5 g, 0.05eq) in benzene (200 mL) was refluxed for 8 h after which water wasremoved using a Dean-Stark apparatus. The cooled solution was dilutedwith diethyl ether (100 mL), washed with aqueous NaOH (15%, 100 mL),brined (100 mL) and dried over Na₂SO₄. After filtration, removal ofsolvent under reduced pressure gave 44 g of crude benzaldehyde(2R)-propylene glycolacetal as an oil.

To a solution of the above crude benzaldehyde (2R)-propyleneglycolacetal (10.0 g, 60.9 mmol) in hexane (100 mL) was addedN-bromosuccinamide (NBS) (11.9 g, 67 mmol, 1.1 eq). The resultingmixture was stirred overnight at room temperature. The suspension wasfiltered through Celite and the filtrate was diluted with hexane (300mL), washed with saturated NaHCO₃ (100 mL) and brine (100 mL), and driedover Na₂SO₄. After filtration, removal of the solvent under reducedpressure gave the title compound 1b (quantitative yield) as an oil. ¹HNMR (400 MHz, CDCl₃): δ 1.48 (d, J=6.4 Hz, 3H), 3.58 (m, 2H), 5.31 (m,1H), 7.43 (t, J=7.6 Hz, 2H), 7.53 (t, J=7.6 Hz, 1H), 8.05 (d, J=7.2 Hz,2H).

Step C: (2R)-2-Phenylcarbonyloxypropyl(2S)-2-(tert-butoxycarbonyl)amino-3-(3,4-dihydroxyphenyl)propanoate (1c)

A suspension of (1R)-2-bromo-1-methylethyl benzoate 1 (4.98 g, 20.6mmol),(2S)-3-(3,4-dihydroxyphenyl)-2-[(tert-butoxycarbonyl)amino]propanoicacid, tetrabutylammonium salt 1a (7.3 g, 25 mmol), and cesiumbicarbonate (4.85 g, 25 mmol) in N,N-dimethylacetamide (100 mL) wasstirred at 55° C. for 16 h. The solvent was evaporated under vacuum.Ethyl acetate was added to the residue and the resulting solution waswashed with water, then 5% NaHCO₃ and brine, and dried over Na₂SO₄.After removing the solvent under reduced pressure, chromatography(silica gel, 30% ethyl acetate in hexane) of the residue gave 6.3 g (68%yield) of the title compound 1c as a white solid. NMR (400 MHz, CD₃OD):δ 1.25 (s, 9H), 1.40 (d, J=6.4 Hz, 3H), 2.99 (dd, J=7.6, 14.4 Hz, 1H),3.10 (dd, J=5.6, 14.4 Hz, 1H), 4.24 (dd, J=5.6, 7.4 Hz, 1H), 4.38 (dd,J=6.8, 11.6 Hz, 1H), 4.52 (dd, J=3.2, 11.6 Hz, 1H), 5.40 (m, 1H), 6.53(dd, J=2.2, 8.4 Hz, 1H), 6.66 (d, J=2.2 Hz, 1H), 6.69 (d, J=8.4 Hz, 1H),7.47 (t, J=7.6 Hz, 2H), 7.60 (t, J=7.6 Hz, 1H), 8.02 (d, J=7.6 Hz, 2H).MS (ESI) m/z 360.15 (M+H)⁺ and 358.09 (M−H)⁻.

Method 1 Step D: (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate hydrochloride (1d)

A solution of (2R)-2-phenylcarbonyloxypropyl(2S)-2-(tert-butoxycarbonyl)amino-3-(3,4-dihydroxyphenyl)propanoate 1c(6.3 g, 13.7 mmol) in 50 mL of 4N HCl in dioxane was stirred at roomtemperature for 30 min. The mixture was concentrated to dryness underreduced pressure. The resulting residue was dissolved in ca. 20 mL ofanhydrous acetonitrile and 4 mL of ether. The solution was refrigerated,and the resulting white precipitate was filtered, washed with ether, anddried under vacuum to afford 4.7 g (87% yield) of the hydrochloride salt1d as a white solid. ¹H NMR (400 MHz, CD₃OD): δ 1.40 (d, J=6.4 Hz, 3H),2.99 (dd, J=7.6, 14.4 Hz, 1H), 3.10 (dd, J=5.6, 14.4 Hz, 1H), 4.24 (dd,J=6, 8 Hz, 1H), 4.38 (dd, J=6.8, 11.6 Hz, 1H), 4.52 (dd, J=3.2, 11.6 Hz,1H), 5.40 (m, 1H), 6.52 (dd, J=2.2, 8.4 Hz, 1H), 6.66 (d, J=2.2 Hz, 1H),6.69 (d, J=8.2 Hz, 1H), 7.47 (t, J=7.6 Hz, 2H), 7.60 (t, J=7.6 Hz, 1H),8.02 (d, J=7.6 Hz, 2H). MS (ESI) m/z 360.15 (M+H)⁺ and 358.09 (M−H)⁻.

Step E: (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, anhydrous (1)

A solution of NaHCO₃ (9.87 g, 117.5 mmol) in water (80 mL) was slowlyadded to a solution of hydrochloride salt 1d (31.0 g, 78.3 mmol) inwater (300 mL). The resulting aqueous suspension was extracted withethyl acetate (EtOAc) (2×400 mL). The combined EtOAc extract was washedwith water, then brine, and dried through MgSO₄. Methanesulfonic acid(6.04 mL, 93.12 mmol) was slowly added to the ethyl acetate (EtOAc)solution while stirred. White precipitate formed as soon as the additionof methanesulfonic acid was complete. The suspension was stirred foranother 30 min and then filtered. The filter cake was washed three timeswith ethyl acetate (EtOAc) and vacuum dried overnight to afford 35.4 g(quantitative yield) of anhydrous mesylate salt 1 as a white solid. ¹HNMR (400 MHz, CD₃OD): δ 1.40 (d, J=6.4 Hz, 3H), 2.70 (s, 3H), 2.98 (dd,J=7.8, 14.6 Hz, 1H), 3.10 (dd, J=5.6, 14.4 Hz, 1H), 4.24 (dd, J=5.8, 7.8Hz, 1H), 4.38 (dd, J=6.8, 12.0 Hz, 1H), 4.52 (dd, J=3.4, 11.8 Hz, 1H),5.40 (dp, J=3.2, 6.4 Hz, 1H), 6.52 (dd, J=2.2, 8.2 Hz, 1H), 6.67 (d,J=2.2 Hz, 1H), 6.69 (d, J=8.0 Hz, 1H), 7.47 (t, J=7.6 Hz, 2H), 7.60 (brt, J=7.4 Hz, 1H), 8.01 (d, J=7.6 Hz, 2H). MS (ESI) m/z 360.07 (M−H)⁺ and358.01 (M−H)⁻.

Method 2

Methanesulfonic acid (3.9 mL, 60.1 mmol) was slowly added to a solutionof (2R)-2-phenylcarbonyloxypropyl(2S)-2-(tert-butoxycarbonyl)amino-3-(3,4-dihydroxyphenyl)propanoate 1c(11.0 g, 22.1 mmol) in 1,4-dioxane (30 mL) while stirred at roomtemperature. The mixture was stirred for 2 h. The solution was slowlyadded to methyl tert-butyl ether (MTBE) (600 mL) with vigorous stirring.The resulting suspension was filtered. The filter cake was washed threetimes with methyl tert-butyl ether and air dried to afford 5.48 g (54%yield) of anhydrous mesylate salt 1 as an off-white solid.

Method 3

A solution of (2R)-2-phenylcarbonyloxypropyl(2S)-2-(tert-butoxycarbonyl)amino-3-(3,4-dihydroxyphenyl)propanoate 1c(10.5 g, 21.1 mmol) in 34 mL (6.0 eq) of 4.0 N HCl/1,4-dioxane wasstirred at room temperature for 1 h. Methanesulfonic acid (1.48 mL, 22.8mmol) was slowly added to the reaction mixture while stirred at roomtemperature. The solution was concentrated under vacuum to afford theanhydrous mesylate salt 1 as a brown solid.

EXAMPLE 2 Preparation of Crystalline Anhydrous(2R)-2-Phenylcarbonyloxypropyl(2S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate (1)

Anhydrous mesylate salt 1 (10.0 g, 22.0 mmol) was dissolved in 200 mL ofisopropanol at 70° C. and the resulting solution was cooled to roomtemperature. Filtration afforded 5.8 g (58% yield) of the crystallineanhydrous mesylate salt 1 as a white crystalline solid (m.p.160.5-161.3° C.). Other solvents and methods useful for crystallizinganhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 are disclosedin Xiang et al., U.S. Pat. No. 7,563,821, which is incorporated byreference its entirety.

EXAMPLE 3 Preparation of Crystalline (2R)-2-Phenylcarbonyloxypropyl(2S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate, Hydrate Method1—High Shear Wet Granulation

Anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (138.6 g) andhydroxypropylmethyl cellulose (1.4 g) (HPMC E4M, Dow Chemical) wereweighed and sieved through an 18-mesh screen. The screened materialswere placed into a high shear wet granulator (KG-5 high sheer blender, 5L bowl, Key International) and pre-blended for 2 min. Water was weighedout (USP, 9.8 g, 7 wt-%). The material was blended for ca. 10-20 min atan impeller speed of 250 rpm and a chopper speed of 2,000 rpm, and awater spray rate of 2 g/min. After granulation the wet granules weremilled through a 16-mesh screen with a brush. The milled wet granuleswere placed in a dryer (UniGlatt Fluid Bed Dryer, Glatt GmbH) and driedfor 24 min at an inlet temperature of 65° C. with airflow adjusted at8-10 SCFM. The dried granules were milled by manually pressing thegranules through a 0.055R screen. As shown by X-ray powder diffractionanalysis, the granules comprised a combination of the anhydrous andhydrated forms of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (FIG. 4).

Using similar processing conditions and 15 wt-% water, the granulescontain predominantly crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate (notshown).

Method 2—Crystallization from Alcohol/Water

Anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (1 g) wassuspended in ca. 1 mL isopropanol/water mixture (40/60 v/v). Thesuspension was stirred at 700 rpm at 25° C. The solid completelydissolved with time (ca. 2 h) and then gradually precipitated out as(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

EXAMPLE 4 X-Ray Powder Diffraction (XRPD) Analysis

X-ray powder diffraction (XRPD) analysis was performed using aPANalytical X'Pert Pro X-ray diffractometer. The X-ray source wasCu-k_(α1) with output voltage of 45 kV and current of 40 mA. Theinstrument employed para-focusing Bragg-Brentano geometry with incidentdivergence and scattering slits set at 1/16° and ⅛°, respectively.0.04-mm radius soller slits were used for both the incident anddiffracted beams to remove axial divergence. Powder samples (9-12 mg)were gently pressed onto a single crystal silicon sample holder to forma smooth surface, and samples were spun at 8 sec/revolution throughoutthe data acquisition process. The samples were scanned from 2° to 40°(2θ°) with a step size of 0.017° (2θ°) and a scan speed of 0.067°/sec.Data acquisition was controlled and analyzed with X'Pert Data CollectorSoftware (version 2.2d) and X'Pert Data Viewer (version 1.2c),respectively.

Representative XRPD diffraction patterns of crystalline anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate and crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate areshown in FIGS. 4, 5, 7, and 8.

EXAMPLE 5 Differential Scanning Calorimetry

Differential scanning calorimetry was performed using a TA InstrumentsQ2000 DSC equipped with a refrigeration cooling system. Samples wereloaded into T_(zero) aluminum pans with crimpled lids. A pinhole wasmade at the center of the lid to prevent pressure buildup duringheating. Following equilibration at 20° C., sample pans were heatedunder a nitrogen atmosphere at a rate of 2° C./min to a finaltemperature of 200° C. Data acquisition was controlled using a ThermalAdvantage Release 4.9.1. The data was analyzed with Universal Analysis2000 software (version 4.5A).

Crystalline anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate prepared fromethanol/water according to Example 2 exhibited a melt transitionbeginning at about 158.0° C., with a peak maximum at about 161.0° C. andhaving a ΔH of about 79.51 J/g (FIG. 2, dashed line).

Crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateprepared from isopropanol/water according to Example 3, Method 2exhibited an endothermic/exothermic transition at about 75° C., which isconsistent with dehydration and recrystallization of anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate (FIG. 2, solidline). At higher temperatures, the anhydrous(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate exhibited a melttransition beginning at about 155.5° C., with a peak maximum at about160.9° C., and having a ΔH of about 61.5 J/g (FIG. 2, dashed line).

EXAMPLE 6 Thermogravimetric Analysis of Crystalline(2R)-2-Phenylcarbonyloxypropyl(2S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate, Hydrate

Thermogravimetric analyses were performed using a TA Instruments Q5000thermogravimetric analyzer equipped with a refrigerated cooling system.Samples were loaded into platinum sample pans, inserted into thethermogravimetric furnace, and heated to 200° C. at a rate of 2°C./minute under a nitrogen atmosphere. Data acquisition was controlledby Thermal Advantage Release 4.9.1. The data was analyzed usingUniversal Analysis 2000 software (version 4.5A).

Thermogravimetric analysis of crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydratecrystallized from propanol/water according to Example 3, Method 2 showedabout 5.5% weight loss from 25° C. to 80° C. The weight loss between 25°C. and 80° C. can be attributed to hydrate water loss: As determined byKarl-Fisher analysis, the water content of the anhydrous form was about0.5 wt-% and the water content of the hydrate was about 5.6 wt-%. Amonohydrate corresponds to a water content of about 3.8 wt-% and adehydrate to a water content of about 7.3 wt-%. A sesqui-hydrate (1.5moles water to 1 mole of compound) corresponds to a water content ofabout 5.6 wt-%.

EXAMPLE 7 Water Sorption/Desorption

The hygroscopicity of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate wasevaluated during one vapor sorption-desorption cycle using a VTI SGA-100dynamic vapor sorption analyzer. (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate wasfirst dried at 25° C. for 180 min under a nitrogen atmosphere. Thematerial was considered equilibrated when the weight change during 2 minwas less than 0.01%. To evaluate the vapor sorption-desorption at 25°C., the dried (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate wasexposed to a humidity cycle ramped (and deramped) from 5% RH to 95% RHat 10% RH intervals. At each humidity interval, the material wasequilibrated as determined by a less than 0.01% weight change during 5min. FIG. 3 shows the humidity-dependent reversible sorption anddesorption of water vapor by (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

EXAMPLE 8 Interconversion of the Anhydrous and Hydrated CrystallineForms of (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate

The anhydrous and hydrated crystalline forms of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate interconvertdepending on the water content of the environment.

When heated from room temperature (25° C.) to 70° C. at a rate of 2°C./min and then held at 70° C. for 15 min, crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate(pattern 1) is converted to a transient/metastable crystalline form(patterns 2 and 3) as shown in FIG. 7. When left at room temperature andhumidity for 1 day, the transient/metastable crystalline form reverts tocrystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate(pattern 4).

When crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate(pattern 2) is vacuum dried overnight and then maintained at roomtemperature and humidity (25° C. and 40-60% RH) for one day, a mixtureof crystalline anhydrous and hydrate forms (pattern 1) are observed asshown in FIG. 8.

As shown in the differential scanning calorimetry scan of FIG. 6, thepropensity of the hydrate to recrystallize can depend on the rate ofdehydration. A DSC of crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate rampedat 2° C./min shows dehydration of the hydrate and recrystallization tothe anhydrous form in the endotherm/exotherm between about 65° C. toabout 80° C., and the melt transition of the anhydrous form beginning atabout 150° C. A DSC ramped at 20° C./min exhibits an endotherm beginningat about 70° C. and without a strong exotherm characteristic ofrecrystallization and without a melt transition at higher temperatures,indicating that the anhydrous form failed to recrystallize under theseconditions.

EXAMPLE 9 Sustained Release Tablet Formulations

Different crystalline forms of a compound can have different solid statephysical properties that can have an impact on the processability of thecompound, the rate of dissolution of the compound from a dosage from,and the stability of the compound. New forms of a pharmaceuticallyuseful compound can provide new opportunities to improve the performancecharacteristics of a pharmaceutical product and can enlarge therepertoire of materials that a formulation scientist has available fordesigning, for example, a pharmaceutical dosage form of a drug with atargeted release profile or other desired characteristics. Crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate can beused to provide oral dosage forms with high drug loading, such as a drugloading of at least about 75 wt-% of the total weight of the oral dosageform.

(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrategranules prepared according to Example 3, Method 2 (100.5 g) weretransferred to a V-shell, 0.5-quart blender, hydroxypropylmethylcellulose (11.3 g) (HPMC K100M, Dow) was added, and the mixture blendedfor 6 min. Magnesium stearate (1.13 g) (NF, Mallinckrodt non-bovine) wassieved through a 20-mesh screen, added to the blender, and the contentsblended for 5 min. Tablets were prepared from the blend using a Betapress equipped with a 5/16-in standard concave tool and pressed to ahardness of 9-15 kP.

Dissolution profiles for tablets were determined using USP Apparatus 2(paddles) at a stirring speed of 50 rpm, a temperature of 37° C.±0.5° C.in 900 mL of 0.1N HCl, pH 1.2. Dissolution profiles for the tabletscontaining 10 wt-% hydroxypropylmethyl cellulose (HPMC K100M, DowChemical) prepared according to the above procedure are shown in FIG. 9.

EXAMPLE 10 Uptake of Levodopa Prodrugs Following Administration ofLevodopa Prodrugs and Carbidopa in Rats

Sustained release oral dosage forms, which release drug slowly overperiods of about 6 to about 24 hours, generally release a significantproportion of the dose within the colon. Thus, drugs suitable for use insuch dosage forms should be colonically absorbed. This experiment wasconducted to assess the uptake and resultant plasma/blood levels oflevodopa, following intracolonic administration of levodopa prodrugmesylate with co-administration of carbidopa (intracolonically,intraperitoneally, or orally), and thereby determine the suitability oflevodopa prodrug mesylate for use in an oral sustained release dosageform. Bioavailability of levodopa following co-administration oflevodopa prodrug mesylate and carbidopa was calculated relative to oralco-administration of levodopa and carbidopa.

Step A: Administration Protocol

Rats were obtained commercially and were pre-cannulated in the both theascending colon and the jugular vein. Animals were conscious at the timeof the experiment. All animals were fasted overnight and until 4 hourspost-dosing of levodopa prodrug. Carbidopa was administered as asolution in water or citrate buffer either orally, intraperitoneally, orintracolonically at a dose equivalent to 25 mg of carbidopa per kganimal weight. Either at the same time or 1 hour after carbidopa dosing,levodopa HCl salt or (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 wasadministered as a solution (in water) directly into the colon via thecannula at a dose equivalent to 75 mg of levodopa per kg. Blood samples(0.3 mL) were obtained from the jugular cannula at intervals over 8hours and were immediately quenched with sodium metabisulfite to preventoxidation of levodopa and levodopa prodrug. Blood was then furtherquenched with methanol/perchloric acid to prevent hydrolysis of thelevodopa prodrug. Blood samples were analyzed as described below.

Step B: Sample Preparation for Colonically Absorbed Drug

Methanol/perchloric acid (300 μL) was added to blank 1.5 mL Eppendorftubes. Rat blood (300 μL) was collected into EDTA tubes containing 75 μLof sodium metabisulfite at different times and vortexed to mix. A fixedvolume of blood (100 μL) was immediately added into the Eppendorf tubeand vortexed to mix. Ten microliters of a levodopa standard stocksolution (0.04, 0.2, 1, 5, 25, and 100 μg/mL) and 10 μL of the 10%sodium metabisulfite solution was added to 80 μL of blank rat blood tomake up calibration standards (0.004, 0.02, 0.1, 0.5, 2.5, and 10μg/mL). Methanol/perchloric acid (300 μL of 50/50) was then added toeach tube followed by the addition of 20 μL of p-chlorophenylalanine.The samples were vortexed and centrifuged at 14,000 rpm for 10 min. Thesupernatant was analyzed by LC/MS/MS.

Step C: LC/MS/MS Analysis

An API 4000 LC/MS/MS spectrometer equipped with Agilent 1100 binarypumps and a CTC HTS-PAL autosampler were used in the analysis. A ZorbaxXDB C8 4.6×150 mm column was used during the analysis. The mobile phaseswere (A) 0.1% formic acid, and (B) acetonitrile with 0.1% formic acid.The gradient condition was: 5% B for 0.5 min, then to 98% B in 3 min,then maintained at 98% B for 2.5 min. The mobile phase was then returnedto 2% B for 2 min. A TurboIonSpray source was used on the API 4000. Theanalysis was done in positive ion mode and the MRM transition for eachanalyte was optimized using standard solution. Five (5) μL of eachsample was injected. Non-compartmental analysis was performed usingWinNonlin software (v.3.1 Professional Version, Pharsight Corporation,Mountain View, Calif.) on individual animal profiles. Summary statisticson major parameter estimates was performed for C_(max) (peak observedconcentration following dosing), T_(max) (time to maximum concentrationis the time at which the peak concentration was observed), AUC_((0-t))(area under the serum concentration-time curve from time zero to lastcollection time, estimated using the log-linear trapezoidal method),AUC_((0-∞))(area under the blood concentration time curve from time zeroto infinity, estimated using the log-linear trapezoidal method to thelast collection time with extrapolation to infinity), and t_(1/2,z)(terminal half-life).

Maximum concentrations of levodopa in the blood (C_(max) values) and thearea under blood concentration versus time curve (AUC) values afterintracolonic dosing of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 with carbidopawere significantly higher (>2-fold) than those achieved for colonicadministration of levodopa with carbidopa.

Intracolonic co-administration of levodopa and carbidopa results in verylow relative bioavailability of levodopa (i.e., only 3% of orallyco-administered levodopa and carbidopa). By comparison,co-administration of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate 1 with carbidopaexhibited improved relative bioavailability of levodopa by at least2-fold. The data demonstrates that certain levodopa prodrugs can beformulated as compositions suitable for effective sustained oral releaseand uptake of levodopa prodrug mesylate and/or levodopa from the colon.

Similar procedures can be used to assess the oral and colonicbioavailability of levodopa administered as(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.

EXAMPLE 11 Use of Crystalline (2R)-2-Phenylcarbonyloxypropyl(2S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate, Hydrate forTreating Parkinson's Disease

The following clinical study may be used to assess the efficacy of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate intreating Parkinson's disease.

Patients with idiopathic PD fulfilling the Queen Square Brain Bankcriteria (Gibb et al., J Neurol Neurosurg Psychiatry 1988, 51, 745-752)with motor fluctuations and a defined short duration levodopa response(1.5-4 hours) are eligible for inclusion. Clinically relevant peak dosedyskinesias following each morning dose of their current medication area further pre-requisite. Patients are also required to have been stableon a fixed dose of treatment for a period of at least one month prior tostarting the study. Patients are excluded if their current drug regimeincludes slow-release formulations of levodopa, COMT inhibitors,selegiline, anticholinergic drugs, or other drugs that could potentiallyinterfere with gastric absorption (e.g. antacids). Other exclusioncriteria include patients with psychotic symptoms or those onantipsychotic treatment patients with clinically relevant cognitiveimpairment, defined as MMS (Mini Mental State) score of less than 24(Folstein et al., J Psychiatr Res 1975, 12, 189-198), risk of pregnancy,Hoehn & Yahr stage 5 in off-status, severe, unstable diabetes mellitus,and medical conditions such as unstable cardiovascular disease ormoderate to severe renal or hepatic impairment. Full blood count, liver,and renal function blood tests are taken at baseline and aftercompletion of the study.

A randomized, double-blind, and cross-over study design are used. Eachpatient is randomized to the order in which either LD/DC or one of thetwo dosages of test compound is administered in a single-dose challengein double-dummy fashion in three consecutive sessions. Randomization isby computer generation of a treatment number allocated to each patientaccording to the order of entry into the study.

Patients are admitted to a hospital for an overnight stay prior toadministration of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate thenext morning on three separate occasions at weekly intervals. Afterwithdrawal of all anti-parkinsonian medication from midnight theprevious day (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate isadministered at exactly the same time in the morning in each patientunder fasting conditions.

Patients are randomized to the order of the days on which they receiveplacebo or (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate. Thepharmacokinetics of crystalline mesylate hydrate may be assessed bymonitoring plasma levodopa concentration over time. Prior toadministration, a 22 G intravenous catheter is inserted in a patient'sforearm. Blood samples of 5 mL each are taken at baseline and at 15, 30,45, 60, 75, 90, 105, 120, 140, 160, 180, 210, and 240 minutes afteradministering (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate oruntil a full off-state has been reached if this occurs earlier than 240minutes after drug ingestion. Samples are centrifuged immediately at theend of each assessment and stored deep frozen until assayed. Plasmalevodopa and 3-O-methyl-Dopa levels are assessed by high-pressure liquidchromatography (HPLC). On the last assessment additional blood may bedrawn for assessment of hematology, blood sugar, liver, and renalfunction.

For clinical assessment, motor function is assessed using UPDRS (UnitedParkinson's Disease Rating Scale) motor score and BrainTest (Giovanni etal., J Neurol Neurosurg Psychiatry 1999, 67, 624-629), which is atapping test performed with a patient's more affected hand on thekeyboard of a laptop computer. These tests are carried out at baselineand then immediately following each blood sampling until the patientreaches their full on-stage, and thereafter at 3 intervals of 20 min,and 30 min intervals until the patient reaches their baselineoff-status. Once a patient reaches their full on-state, video recordingsare performed three times at 20 min intervals. The following mental andmotor tasks, which have been shown to increase dyskinesia (Duriff etal., Mov Disord 1999, 14, 242-245), are monitored during each videosession: (1) sitting still for 1 minute; (2) performing mentalcalculations; (3) putting on and buttoning a coat; (4) picking up anddrinking from a cup of water; and (5) walking. Videotapes are scoredusing, for example, versions of the Goetz Rating Scale and the AbnormalInvoluntary Movements Scale to document a possible increase in testcompound induced dyskinesia.

Actual occurrence and severity of dyskinesia can be measured with aDyskinesia Monitor (Manson et al., J Neurol Neurosurg Psychiatry 2000,68, 196-201). The device is taped to a patient's shoulder on their moreaffected side. The monitor records during the entire time of achallenging session and provides a measure of the frequency and severityof occurring dyskinesias.

Results can be analyzed using appropriate statistical methods.

EXAMPLE 12 Clinical Trial to Determine the Pharmacokinetics and Safetyof (2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate Mesylate, Hydrate

An open-label, single-center, randomized, 3-treatment, 3-period,single-dose clinical study can be used to assess the PK and safety ofpharmaceutical compositions comprising of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate(administered with Lodosyn® (carbidopa)) compared with Sinemet® (L-dopaand carbidopa) in healthy adult subjects. Treatments are administered inboth fasted and fed conditions.

Prior to initial dosing on Day −1 of Period 1, 24 subjects arerandomized to one of two parallel groups using a 1:1 allocation.Subjects assigned to Group 1 are randomized to one of two 2-periodsequences (AB or BA) to examine formulation effect in the fasted state.Subjects assigned to Group 2 are randomized to one of two 2-periodsequences (AB or BA) to examine formulation effect in the fed state.Subjects will receive Treatment C in the third period in either thefasted (Group 1) or the fed (Group 2) state. Subjects will also receive50 mg (2×25 mg) Lodosyn® tablets at −24, −12, 12 and 24 hours postTreatments A-C.

Subjects in Group 1 (Fasted) receive Treatment A: 2×SR2 Tablets(sustained release tablet 2 comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate)+50 mg(2×25 mg) Lodosyn® Tablets; Treatment B: 2×SR3 Tablets (sustainedrelease tablet 3 comprising (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate)+50 mg(2×25 mg) Lodosyn® Tablets; and Treatment C: 200 mg (2×25/100 mg)Sinemet® Tablets. Subjects in treatment Group 2 (Fed) receive TreatmentA: 2×SR2 Tablets+50 mg (2×25 mg) Lodosyn® Tablets; Treatment B: 2×SR3Tablets+50 mg (2×25 mg) Lodosyn® Tablets; and Treatment C: 200 mg(2×25/100 mg) Sinemet® Tablets.

Subjects begin screening within 16 days of checking into the clinic forPeriod 1. For Period 1-3, subjects check into the clinic on Day −2, andremain on site until check out (Day 2, 36 hours post-dose) after thelast blood draw. Periods 1 through 3 last approximately 3 weeks, duringwhich subjects are confined to the clinic for approximately 4 days (Day−2 thru Day 2) during each Period, for a total of 12 days ofconfinement. Following discharge from Periods 1 and 2, subjects completea washout period of approximately 1 week, which starts on Day 1 afteradministration of the morning Treatment. A final follow-up visit occurs5-7 days after clinic discharge for Period 3.

All subjects fast overnight on Day −1 for approximately 10 hours. Fastedtreatments (Treatments A, B and C for Group 1) are administeredfollowing the 10-hour overnight fast. Fed treatments (Treatments A, B,and C for Group 2) are administered within 10 minutes of completion of ahigh fat breakfast (ca. 50% calories from fat; ca. 800-1000 totalkcals). Lodosyn® tablets at −24, −12 (pre-dose to Day 1), 12, and 24hours post Treatments A-C are administered following a moderate fatmeal.

Blood and urine are collected at intervals up to 36 hours post-dosebeginning on Day 1 of each period.

On Day 1, subjects dosing under fed conditions are served a high-fat(ca.50 percent of total caloric content of the meal) and high-calorie(ca. 800 to 1000 calories) meal for breakfast (e.g., 2 eggs fried inbutter, 2 strips bacon, 2 slices of wheat toast with butter, 4 ounces ofhash brown potatoes, and 9 ounces of whole milk), ca. 30 minutes priorto dosing. The test meal is to derive approximately 150, 250, and500-600 calories from protein, carbohydrate, and fat, respectively.Subjects must consume the entire meal. Within 10 minutes of consumptionof the high fat breakfast, subjects receive the dose of the study drugwith 240 mL of non-carbonated water. All other meals (dinner on Day −2,breakfast, lunch, and dinner on Day −1, lunch and dinner of Day 1 andbreakfast, lunch and dinner on Day 2) are standardized clinic meals(approximately 30% calories from fat). Subjects may be served anoptional snack after dinner.

Blood (4 mL per sample) is taken from subjects for estimation ofpharmacokinetic parameters. Blood samples are quenched for determinationof concentrations of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate, levodopa,3-O-methyldopa, carbidopa, and other metabolites. The following PKparameters are estimated using non-compartmental methods: peakconcentration (C_(max)), time to peak concentration (T_(max)),concentration at 12 hr post-dose (C₁₂), terminal elimination rateconstant (Kel) and half-life (T_(1/2)), and area under theconcentration-time curve (AUC).

Urine is collected and analyzed for determination of concentrations oflevodopa and other metabolites. The following are estimated: the amountof the analyte excreted in urine during each collection interval(Ae_((0-t))), the amount of the analyte excreted in urine during the36-hour post-dose interval (Ae₍₀₋₃₆₎), and the cumulative percentage ofdose excreted in urine up to 36 hours post-dose (% dose excreted).

Data from all study periods can be analyzed using appropriatestatistical methods.

EXAMPLE 13 Crystal Structures of Anhydrous and Hydrate Forms of(2R)-2-Phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate

Crystal structures of anhydrous and hydrate forms were determined toevaluate the molecular structures, conformations, overall packing andarrangements of groups at the possible crystal faces. The X-ray data wascollected at −173° C. The anhydrous form of(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate crystallizes inthe chiral space group P2₁ (Table 1) with one molecule in the asymmetricunit. The structure of the asymmetric unit with the conformation of thedrug molecule is shown in FIG. 10. The mesylate anion and the —NH₃ ⁺group of the drug are connected to each other through strongcharge-assisted N—H . . . O hydrogen bonds. In addition, the hydroxylgroups of the catechol moiety are bonded to the mesylate anion and theester carbonyl groups through N—H . . . O hydrogen bonds. This packingleads to an overall layered structure with distinct hydrogen bondingdomains and domains containing aromatic groups (FIG. 11). The structureexhibits a packing fraction of 0.681 and inter-connected cavities downthe b-axis in the vicinity of aromatic domains.

TABLE 1 Crystal data for anhydrous levodopa prodrug mesylate. FormulaC₁₉H₂₂NO₆:CH₃O₃S Crystal System Monoclinic Space group P2₁ (No. 4) a, b,c [Å] 10.4410(4), 5.9120(2), 17.6530(8) α, β, γ[°] 90, 94.826(2), 90 V[Å³] 1085.81(7) Z 2 D_(calc) [g cm⁻³] 1.393

The hydrate form of (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate alsocrystallizes in the chiral space group P2₁ (Table 2). The latticestructure contains two symmetry independent mesylate salt species andthree symmetry independent water molecules, giving an overall 1:1.5stoichiometry between the compound and water. The structure of theasymmetric unit with the conformations and some inter-component hydrogenbonds is shown in FIG. 12. While independent(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate molecules stilladopt bent conformations, the conformation in the anhydrous form is morecompact compared to the ones in the hydrate. As in the anhydrous form,the —NH₃ ⁺ and —OH groups participate in hydrogen bonding. In thehydrated form, however, the hydrogen bond pattern is different due tothe insertion of water molecules in the hydrogen bond network. Thehydrate also forms a layered structure (FIG. 13) with polar domainscontaining charged groups and hydrogen bonds and aromatic domainscontaining phenyl groups. The structure of the hydrate form has slightlyhigher density (1.411 g cm⁻³) and packing fraction (69.7%) compared tothe anhydrous form. The slightly enlarged conformation of the moleculesand added water molecules in the lattice lead to a larger d-spacing forthe interlayer distance in the hydrate.

TABLE 2 Crystal data for levodopa prodrug mesylate, hydrate. Formula2(C₁₉H₂₂NO₆):2(CH₃O₃S):3H₂O Crystal System Monoclinic Space group P2₁(No. 4) a, b, c [Å] 8.1519(1), 29.5903(4), 9.4261(1) α, β, γ[°] 90,92.415(1), 90 V [Å³] 2271.72(5) Z 2 D_(calc) [g cm⁻³] 1.411

Finally, it should be noted that there are alternative ways ofimplementing the embodiments disclosed herein. Accordingly, the presentembodiments are to be considered as illustrative and not restrictive.Furthermore, the claims are not to be limited to the details givenherein, and are entitled their full scope and equivalents thereof.

1. A compound crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate, whichexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 14.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, and 18.5°±0.2° in anX-ray powder diffraction pattern measured using Cu—K_(α) radiation. 2.The compound of claim 1, which exhibits characteristic scattering angles(2θ) at least at 6.0°±0.2°, 9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°,13.8°±0.2°, 14.6°±0.2°, 15.1°±0.2°, 15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°,17.6°±0.2°, 18.5°±0.2°, and 19.2°±0.2° in an X-ray powder diffractionpattern measured using Cu—K_(α) radiation.
 3. The compound of claim 1,which exhibits characteristic scattering angles (2θ) at least at6.0°±0.2°, 9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°, 14.6°±0.2°,15.1°±0.2°, 15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°, 18.5°±0.2°,19.2°±0.2°, 20.8°±0.2°, 21.9°±0.2°, 22.8°±0.2°, 23.4°±0.2°, 23.7°±0.2°,23.9°±0.2°, and 26.5°±0.2° in an X-ray powder diffraction patternmeasured using Cu—K_(α) radiation.
 4. The compound of claim 1, whichexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 9.6°±0.2°, 11.2°±0.2°, 12.0°±0.2°, 12.8°±0.2°, 13.8°±0.2°,14.3°±0.2°, 14.6°±0.2°, 15.1°±0.2°, 15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°,17.6°±0.2°, 18.5°±0.2°, 18.7°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 20.8°±0.2°,21.1°±0.2°, 21.9°±0.2°, 22.8°±0.2°, 23.4°±0.2°, 23.7°±0.2°, 23.9°±0.2°,24.7°±0.2°, 26.5°±0.2°, 28.2°±0.2°, 28.3°±0.2°, and 29.5°±0.2° in anX-ray powder diffraction pattern measured using Cu—K_(α) radiation. 5.The compound of claim 1, comprising from about 1.4 moles water to about1.6 moles water to mole of the compound.
 6. The compound of claim 1,comprising from about 5.3 wt-% to about 5.7 wt-% water.
 7. The compoundof claim 1, wherein the compound is prepared by a process comprisingprocessing (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate by high shearwet granulation using a water content ranging from about 10 wt-% toabout 20 wt-% to provide granules comprising the crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.
 8. Thecompound of claim 1, wherein the compound is prepared by a processcomprising: dissolving (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate in a solventcomprising water to form a solution, wherein the water activity in thesolvent is greater than about 0.6; and crystallizing the(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate to provide thecrystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate.
 9. Thecompound of claim 8, wherein the solvent comprises isopropanol and atleast about 7%-bv water.
 10. A pharmaceutical composition comprisingcrystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate, whichexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 14.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, and 18.5°±0.2° in anX-ray powder diffraction pattern measured using Cu—K_(α) radiation and apharmaceutically acceptable vehicle.
 11. The pharmaceutical compositionof claim 10, comprising a therapeutically effective amount of thecrystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate fortreating a disease in a patient, wherein the disease is chosen fromParkinson's disease, depression, attention deficit disorder,schizophrenia, manic depression, cognitive impairment disorders,restless legs syndrome, periodic limb movement disorders, tardivedyskinesia, Huntington's disease, Tourette's syndrome, hypertension,addictive disorders, congestive heart failure, and excessive daytimesleepiness.
 12. The pharmaceutical composition of claim 11, wherein thedisease is Parkinson's disease.
 13. The pharmaceutical composition ofclaim 10, comprising an L-aromatic amino acid decarboxylase inhibitor.14. The pharmaceutical composition of claim 10, comprising acatechol-O-methyltransferase inhibitor.
 15. The pharmaceuticalcomposition of claim 10, formulated for sustained release oraladministration.
 16. An oral dosage form comprising crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate, whichexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 14.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, and 18.5°±0.2° in anX-ray powder diffraction pattern measured using Cu—K_(α) radiation. 17.The oral dosage form of claim 16, wherein the amount of the crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate in theoral dosage form is at least about 75 wt-%.
 18. The oral dosage form ofclaim 16, further comprising anhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate.
 19. The oraldosage form of claim 18, wherein the combined amount of the crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate and theanhydrous (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate in the oraldosage form is at least about 75 wt-%.
 20. The oral dosage form of claim16, which is a sustained release formulation.
 21. The pharmaceuticalcomposition of claim 10, wherein the crystalline(2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°, 14.6°±0.2°, 15.1°±0.2°,15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°, 18.5°±0.2°, and19.2°±0.2° in an X-ray powder diffraction pattern measured usingCu—K_(α) radiation.
 22. The pharmaceutical composition of claim 10,wherein the crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°, 14.6°±0.2°, 15.1°±0.2°,15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°, 18.5°±0.2°, 19.2°±0.2°,20.8°±0.2°, 21.9°±0.2°, 22.8°±0.2°, 23.4°±0.2°, 23.7°±0.2°, 23.9°±0.2°,and 26.5°±0.2° in an X-ray powder diffraction pattern measured usingCu—K_(α) radiation.
 23. The oral dosage form of claim 16, wherein thecrystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrate (2θ) atleast at 6.0°±0.2°, 9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°,14.6°±0.2°, 15.1°±0.2°, 15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°,18.5°±0.2°, and 19.2°±0.2° in an X-ray powder diffraction patternmeasured using Cu—K_(α) radiation.
 24. The oral dosage form of claim 16,wherein the crystalline (2R)-2-phenylcarbonyloxypropyl(2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate mesylate, hydrateexhibits characteristic scattering angles (2θ) at least at 6.0°±0.2°,9.1°±0.2°, 9.6°±0.2°, 12.0°±0.2°, 13.8°±0.2°, 14.6°±0.2°, 15.1°±0.2°,15.6°±0.2°, 16.1°±0.2°, 16.6°±0.2°, 17.6°±0.2°, 18.5°±0.2°, 19.2°±0.2°,20.8°±0.2°, 21.9°±0.2°, 22.8°±0.2°, 23.4°±0.2°, 23.7°±0.2°, 23.9°±0.2°,and 26.5°±0.2° in an X-ray powder diffraction pattern measured usingCu—K_(α) radiation.